Alginate cross-linked with divalent metal ions has been used for microencapsulation of cells, bacteria, drugs and dyes for several decades. Divalent calcium ions (Ca2+) are widely used to cross-link alginate. The most common encapsulation techniques are extrusion and emulsification/gelation using alginate as a support material (Fundueanu 1998, Krasaekoopt 2003, Yu 2008). Depending on the source of hardening ions used to cross-link alginate, emulsification/gelation methods are further divided into “internal”, in which insoluble calcium salt is mixed with alginate and solubilized after emulsification by change of pH and “external”, in which soluble calcium salt is dropped into an emulsion containing alginate (Poncelet 1992, Chan 2001b, Chan 2005, Ribeiro 2005, Ching 2008, Song 2013).
Other divalent metal ions (Zn2+, Sr2+ and Ba2+) can bind and cross-link alginate carboxylic groups in a stronger and less selective manner (Gray 1987, Aslani 1996). However, barium and strontium are not approved for food and feed applications.
Microcapsules cross-linked only with zinc ions are said to aggregate and create clumps of much higher diameters than expected. Therefore, cross-linking is performed using combinations of Zn2+ and Ca2+ ions, or solely with Ca2+ ions (Chan 2001a).
Gray (1987) described microencapsulation of insulin. Zinc ions bind alginate less selectively than calcium ions, resulting in smaller pores in zinc alginate matrices. However, no significant difference was observed between Zn2+ and Ca2+ ions. A high retention of insulin in zinc alginate was attributed to insulin binding with zinc.
Poncelet (1992) described an emulsification/internal gelation technique using a calcium alginate gel for microencapsulation of bacteria.
Smit (1995) described microencapsulation of bacteria to protect bacteria from bacteriophage using an extrusion technique using calcium and alginate.
Aslani (1996) observed that zinc binds alginate less selectively, providing a denser matrix, so the release from zinc alginate was retarded when compared to calcium alginate. However, the main conclusion was that there were no particular advantages of using zinc ions instead of calcium ions.
Fundueanu (1998) compared extrusion and emulsification techniques of microencapsulation with calcium alginate.
Chan (2001) compared alginate cross-linking methods using Ca2+. Zn2+ and combinations of both, concluding that a combination of both ions gave the best results.
Chan (2001) compared microencapsulation via external and internal gelation methods. An internal calcium source was favoured, despite this providing bigger pores in the microcapsules, because external calcium addition caused the emulsion to break, resulting in the microcapsules forming clumps.
Shu (2002) described microencapsulation via an extrusion technique, using alginate, Ca2+ ions and chitosan. Calcium ions and chitosan were combined in a single solution for alginate hardening.
Krasaekoopt (2003) compared methods of microencapsulation of probiotics using extrusion and emulsification techniques with calcium ions and alginate.
Chan (2005) compared microencapsulation using external and internal gelation methods, concluding that external gelation resulted in microcapsules with smoother membranes and smaller pores.
Ribeiro (2005) described an emulsification/internal gelation method for microencapsulation of haemoglobin, in which alginate was cross-linked using calcium ions and was coated with chitosan.
Xu (2006) described a method in which an alginate solution was mixed with chitosan powder and extruded into a Ca2+ solution, chitosan was then dissolved by pH adjustment.
Ching (2008) described a method in which microcapsules were produced via external gelation; alginate was cross-linked using Ca2+ salts of different solubilities.
Ma (2008) described microencapsulation of Salmonella bacteriophage Felix O1 in calcium alginate capsules via extrusion techniques, which were coated with chitosan.
Yu (2008) described microencapsulation using an extrusion technique. Alginate was extruded into a mixture of calcium ions and chitosan in a single-step hardening process.
Puapermpoonsiri (2009) described a water/oil/water (w/o/w) double emulsion/solvent extraction technique, using PLGA, PVA and gelatin to microencapsulate S. aureus and P. aeruginosa bacteriophage. The microcapsules produced were freeze dried successfully.
Ma (2010) described microencapsulation of S. aureus bacteriophage K in calcium alginate capsules coated with chitosan via extrusion techniques. Lyophilisation of the product required addition of sugars, such as trehalose.
Song (2013) compared emulsification/external and internal gelation methods for microencapsulation of probiotics using a calcium alginate matrix coated with chitosan.
US 2012/0263826 A1 describes comestible products containing encapsulated probiotic bacteria having resistance to thermal and acidic conditions; methods for encapsulation of probiotics are described in which a mixture of denatured protein and sodium alginate (in a ratio of from 1:1 to 1:9) with active probiotic cells is combined with a divalent cation, specifically Ca2+, to initiate cold gelation of the sodium alginate and protein to form a second mixture which is then extruded through an opening of diameter less than 1000 μm to form capsules.
EP 1537860 A1 describes a vaccine composition and a method of preparation including the steps of: forming a water-in-oil emulsion including an alginate in water, an oil, an antigen, and at least one of (a) a cellulose ether and at least one non-ionic surfactant; and (b) a poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) tri-block copolymer surfactant and at least one non-ionic surfactant; followed by crosslinking the alginate in the emulsion with at least two cations selected from the group consisting of aluminium, barium, calcium, lithium, manganese, strontium, and zinc, to form antigen-containing, cross-linked alginate microparticles; and harvesting the microparticles.
WO 2009/037264 A2 describes antimicrobial compositions of bacteriophage, phage proteins, antimicrobial peptides, or antimicrobial aptamers for oral delivery, adapted for the delivery of the active to the colon, distal ileum, or other portion of the gastrointestinal tract other than the stomach. The compositions can include pectin beads formed by cross-linking pectin with zinc or any divalent, trivalent, or polycationic cation: optionally the pectin beads can be coated with a polycationic polymer, and/or coated with any suitable polymer for targeted delivery of the active ingredient to the desired part of the gastro-intestinal tract such as Eudragit®-type polymers.
It is an object of the invention to provide improved methods for microencapsulation, microcapsules obtained by such improved methods and uses of such microcapsules.